Bifunctional energy-reversible acyl-compositions

ABSTRACT

Energy-reversible acyl conjugates, intermediates, and related compositions are disclosed. In preferred aspects, examples of such compositions include:

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/324,053filed Dec. 30, 2005, which is a divisional of U.S. patent applicationSer. No. 10/066,323 filed Jan. 31, 2002, now U.S. Pat. No. 7,157,458which, in turn, claims the benefit of priority from U.S. ProvisionalPatent Application No. 60/284,308, filed Apr. 17, 2001, the contents ofeach of which are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present application relates generally to energy reversibleacyl-enzymes. In particular, the present application relates to energyreversible acyl-enzymes and the like having a cinnamate or relatedstructure core and an additional reactive group which can be modified toimpart new properties to the whole composition.

Various methods to make use of enzyme inactivation have been disclosed.Enzyme inhibition can be used to enhance long term storage of enzymes orto inactivate the enzymes in a pharmaceutical drug. For example, U.S.Pat. No. 5,770,699 describes a process of enzyme inhibition to produceinactivated blood factors. U.S. Pat. No. 5,837,679 discloses a method toextend the half lives of blood factors via a transient modification ofblood factors by acylation. U.S. Pat. No. 4,337,244 reports a method oftreating venous thrombosis using an inactivated fibrinolytic enzyme.

Enzyme activity can be controlled with inhibitors. Reversible control ofenzyme activity with light has been the focus of a number of reports(see U.S. Pat. Nos. 5,114,851 and 5,218,137 to Porter et al.). There area number of advantages of this concept. Most stricing is the ability tocontrol enzyme activity specifically and rapidly, by exposure to lightin vivo or ex vivo.

Porter et al. disclose in U.S. Pat. Nos. 5,114,851 and 5,218,137, thelight controllable enzymes are obtained by coupling an enzyme activesite amino acid residue to cinnamate (CINN) derivatives to formo-hydroxy cinnamate substituted esters or acyl enzymes, which areinactive. On photolysis, the bond with the active site amino acidresidue is cleaved and the active site is exposed. Pizzo et al. [(1986)Ann. N.Y. Acad. Sci. 485: 199-203] reported on the use of an o-hydroxycinnamate substituted ester, formed by coupling the active site of anenzyme to α-methyl-2-hydroxy-4-diethylaminocinnamic acid.

A subsequent report by Porter, et al. demonstrated the therapeuticpotential of the inhibited enzymes. Control of clotting reaction timeswas concentration dependent and photolysis time dependent. In vivoclotting of abnormal blood vessels in a rabbit model of cornealneovascularization was achieved by injection of the inhibited, “caged”enzymes and application of 366 nm light to the eyes for 25 minutes. SeeArroyo et al., Thromb. Haemost. 78, 791-793 (1997). U.S. Pat. Nos.5,114,851 and 5,218,137, further describe these cinnamate derivatizedenzymes and uses thereof.

The usefulness of the inhibited enzymes is a function of the rate andextent of photolysis. Utility is limited if rate of bond cleavagebetween the cirtamate moiety and the active-site amino acid is slow.Many applications require rapid exposure times, on the order of secondsin most in vitro or typical applications, or at most minutes, in most invivo applications. Rapid, controlled response times are essential formost clinical applications and are of particular importance with labileenzymes or uses where rapid reaction times are essential, as inclotting.

For example, the formation of an acyl-enzyme between α-chymotrypsin andthe p-nitrophenyl ester of p-nitro-trans-cinnamic acid is described byVarfolomeyev, S., et al., [FEBS Lett. 15:118 (1971)]. The bond betweenthe enzyme and the carboxylate group is formed with the hydroxyl groupof the serine at the catalytic center of the enzyme [Berezin, I. et al.,FEBS Lett. 8:173 (1970)]. The formation of an acyl-enzyme betweena-thrombin and the trans-isomer of an ester ofo-hydroxy-a-methylcinnamic acid is described by Turner, A., et al., J.Amer. Chem. Soc.109:1274 (1987). The bond between the enzyme and thecarboxylate group is formed with the hydroxyl group of the serine-195 atthe catalytic center of the enzyme (Turner, A., et al., J. Amer. Chem.Soc. 110:244 (1988)). Exposure of the compound to light led todeacylation. The photoactivation of these enzymes was slow and requiredlight intensities and wavelengths such that appreciable enzymedegradation occurred during photoactivation.

Research in this area has continued. For example, Porter, et al.(Photochem. Photobiol. B 38(1), 61-69 (1997) inserted a biotinderivative on the 2-position of the cinnamate side chain (adjacent tothe carboxylate group), which could be bound to avidin, for purposes ofpurification and immobilization. The modified compound also maintainedthe ability to be photoactivatable.

Further modifications of the CINN core molecule would be desirable inorder to improve inhibited enzyme compositions that can be rapidly andcontrollably reactivated. For example, it would be desirable tointroduce additional sites to react with other molecules for purposes ofchanging the properties of the acyl-enzyme (such as immobilization orpharmacokinetics), while maintaining the desired properties ofphoto-activation. The present invention addresses this need.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide improvedcompositions designated herein as Z-CINN-X₁-A, including inhibitedenzymes that can be rapidly and controllably reactivated.

It is another object of the present invention to provide an additionalsite on Z-CINN-X₁-A type compounds (as defined below) which can bederivatized in various ways, including with groups designated herein as“B-L”, to provide compositions designated herein as B-L-Z-CINN-X₁-A,with additional properties such as stability, targeting capacity, orimmobilization to appropriate supports.

It is yet another object of the present invention to provide an enzymeinhibitor composition (B-L-Z-CINN-X₁-A₁), which is capable of generatingan inactivated enzyme composition (B-L-Z-CINN-X₁-A₂), where the enzymecan be released in active form via a controlled mechanism such as inputof light energy.

These and other objects are provided by the present invention which inone embodiment provides compositions corresponding to Z-CINN-X₁-A andFormula (I):

wherein:

R₁ and R₂ are individually selected from among H, CH₃, C₂-C₁₀alkyls,alkenyls or alkynyls which can be substituted or unsubstituted; straightor branched; C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroajkynyls and—(CR₁₅R₁₆)_(p)-D,

-   -   wherein: R₁₅ and R₁₆ are individually selected from the group        consisting of H, CH₃, C₂-C₁₀ alkyls, alkenyls or alkynyls which        can be substituted or unsubstituted; straight or branched;        C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls;    -   p is a positive integer from 1 to about 12;    -   D is selected from among —SH, —OH, X₂, —CN, —OR₁₉, NHR₂₀,

-   -   -   wherein:        -   R₁₇ is H, CH₃ or X₃;        -   R₁₈ is H, a C₁-C₄ alkyl or benzyl;        -   R₁₉ is H, a C₁₋₄ alkyl, X₂ or benzyl;        -   R₂₀ is H, a C₁₋₁₀ alkyl or —C(O)R₂₁,            -   wherein R₂₁ is H, a C₁₋₄ alkyl or alkoxy, t-butoxy or                benzyloxy;        -   X₂ and X₃ are independently selected halogens;

    -   R₃ is H, CH₃, or —C(═O)(CR₁₅R₁₆)_(w)-D,

    -   where w is 0 or an integer from 1 to about 12, and D is H or as        described for R₁ and R₂.

    -   J is O NH or S;

    -   R₄, R₅, and R₆ are independently selected from among H, CH₃,        C₂-C₁₀ alkyls, alkenyls or alkynyls which can be substituted or        unsubstituted; straight or branched; C₂-C₁₀ heteroalkyls,        heteroalkenyls or heteroalkynyls and halogens;

Z is NR₇R₈ or

wherein R₇ is selected from among H, CH₃, C₂-C₁₀ alkyls, alkenyls oralkynyls which can be substituted or unsubstituted; straight orbranched; C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls, or(CR₂₃R₂₄)_(q)-aryl, or R₈;

wherein R₂₃ and R₂₄ are independently H or a C₁-C₁₀ alkyl;

q is an integer from 1 to about 6;

R₈ is selected from among (CR₉R₁₀)_(n)—NR₂₂—R₁₁,(CR₉R₁₀)_(n)—CH₂—NHC(O)R₂₆ and (CR₉R₁₀)—CH₂-E,

wherein R₉ and R₁₀ are independently selected from among H, CH₃, C₂-C₁₀alkyls, alkenyls or alkyls which can be substituted or unsubstituted;straight or branched;

C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls and halogens;

R₂₆ is A, CH₃, O-t-butyl, O-benzyl;

E is OH, SH or O—C(O)R₂₇,

-   -   wherein R₂₇ is a C₁-C₆ alkyl, benzyl or phenyl;

R₂₂ is H or CH₃;

n is a positive integer from 1 to about 10;

R₁₁, is H or -L-B

-   -   wherein L is a linker; and        -   B is a first active moiety, containing reactive group            moieties such as maleimidyl or N-hydroxysuccinimidyl, etc.,            or a polymer;

R₂₅ is H, —C(O)—R₂₈ or —C(O)—O—R₂₉,

-   -   wherein R₂₈ is a C₁-C₆ alkyl or benzyl; and R₂₉ is CH₃, t-butyl        or benzyl;

X₁ is O, NH, or S; and

A is H or a second active moiety, designated as either A₁ or A₂ herein,it being understood that X₁ is an integral part of either A₁ or A₂.

Pharmaceutically acceptable salts, including Cl⁻, Br⁻, HSO⁻⁴, etc. ofcompounds corresponding to Formula (I) are also provided.

In preferred aspects, B is a polymer or natural or synthetic organicmolecule, a protein, such as an antibody or fragment thereof acarbohydrate, a nucleic acid, a lipid, or other naturally occurring orsynthetic compounds.

In further preferred embodiments, A is A₁, and X₁A₁ is a substrate orsubstrate analog such as an amino acid, an amino acid derivative, apeptide, a peptide derivative or a substrate or substrate analog forserine proteases, cysteine proteases, esterases, lipases, or otherenzymes containing an active site serine or cysteine. In some preferredaspects, X₁A₁ is:

in which R₁₂ and R₁₃ are H or electron donating or electron withdrawinggroups and W is a covalent bond. Alternatively, A₁ can also be an aminoacid or amino acid derivative, peptide or peptide derivative or othermolecule which is a substrate or substrate analog for serine proteases,cysteine proteases, esterases, lipases, or other enzymes containing anactive site serine or cysteine.

In other aspects of the invention, H—X₁A₂ is an enzyme such as a serineprotease, cysteine protease, esterase, lipase, or other enzymecontaining an active-site serine, cysteine or tysine whose side-chain—O, —S or —NH corresponds to X₁ of Formula (I) which is bonded to theC(O) of the Z-CINN upon displacement of X₁A₁. Thus, H—X₁A₂ is an enzymerendered inactive through its bond to Z-CINN and is preferably capableof having its enzyme activity restored by hydrolysis or by exposure tolight or other energy source.

In still further aspects of the invention, Z-CINN-X₁-A₁ compositions arederivatized utilizing the reactive Z site of Formula I. In particular,when R₈ is (CR₉R₁₀)—NR₂₂—R₁₁, and R₁₁ is L-B, the artisan is providedwith Z-CINN cores which are linked to, among other things, polymers suchas PEG or other activated polymers.

Alternatively, the Z-CINN-X₁-A can be joined to a linker group L so thatsubsequent attachment of B groups, e.g. monoclonal antibodies (mAbs),polymers, etc. can be carried out when desired. The Z-CINN-X₁-A can belinked using, for example,succinimidyl-6-[(β-maleimidopropionamido)hexanoate], ethylene glycolbis[succinimidyl succinate], bis activated PEGs containing terminalsuccinimidyl succinates, N-hydroxy-succinimidyls and/or maleimides.Other bifunctional reagents are also contemplated. The L-Z-CINN-X₁-Amolecules can then be reacted with another molecule, e.g. proteins,including antibodies, fragments thereof, nucleic acids, lipids, or othernaturally occurring or synthetic compounds. Derivatized compositions(B-L-Z-CINN-X₁-A₁) can then be reacted with an enzyme to formB-L-Z-CINN-X-A₂, where the enzyme is inactivated.

As a result of the present invention, several advantages are provided.For example, these Z-CINN-X₁-A inactivated compositions can have variousbeneficial properties, such as increased solubility, increased half-lifein circulation, targeting ability, or other features. In addition,L-Z-CINN-X₁-A inactivated compositions can be immobilized bycrosslinking to support materials via the linker L. The supportmaterials can be any industrially or pharmaceutically suitable materialssuch as organic polymers, inorganic polymers, natural polymers,biopolymers or zeolites and can be in the form of films, membranes,filters, beads, particles, resins, microparticles, or columns.Alternatively, B-L-Z-CINN-X₁-A can be attached to supports by mechanismssuch as affinity or by additional coupling reactions with activatedsupport materials.

The acyl-enzyme bonds can be relatively stable or susceptible tohydrolysis at about neutral pH in the dark. They are susceptible tocleavage by a source of energy such as light, including ultraviolet,visible, and infrared light, microwave, ultrasound, radiowave energy orradioactivity. The preferred energy source is light having a wavelengthin the range from 340 to 700 nm and preferably 350 to 420 nm. Thisprovides, for example, a means to controllably release and convertinactivated, acylated enzymes to active enzymes.

Compounds of the invention corresponding to, for example,B-L-Z-CINN-X₁-A₂, can be used in an assay, where the acyl-enzyme isfirst free or bound to an immobilized support, then energy is applied torelease the active enzyme into solution. The inactivated compositionscan also be used in purification methods, and as therapeutics, which areactivated at the time of or shortly before administration. Theinactivated enzymes can be stored for lengthy periods, then reactivatedat time of use, to increase shelf lives.

In further aspects of the invention, methods of preparing and using thecompositions of the present invention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the synthesis of(3-[2-hydroxy-4-(ethyl)(2-tert-butylcarbamidoethyl)amino]phenyl-2-methyl-2-propenoicacid, ethyl ester) (10).

FIG. 2 illustrates the synthesis of(3-[2-hydroxy-4-(ethyl)aminoethyl)-amino]phenyl-2-methyl-2-propenoicacid, 4-aminoiminophenyl ester, HCl salt) (N-CINN-AP) (16).

FIG. 3 illustrates the preparation of mPEG₅₀₀₀-N-CINN-AP (18).

FIG. 4 illustrates the preparation of derivatized inhibited enzyme (19)by reaction of mnPEG₅₀₀₀-N-CINN-AP (18) and enzyme.

FIG. 5 illustrates the preparation of a maleimido derivative ofN-CINN-AP (20), Mal-L-N-CINN-AP, by reaction of (16) withsuccinimidyl-6-[(β-rnaleimidopropion-amido)hexanoate].

FIG. 6 illustrates the preparation of antibody-L-N-CINN-AP (21) byreaction of SH-modified protein with (20), followed by reaction withenzyme to give antibody-Mal-L-N-CINN-enzyme (22).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “inhibitor” as used herein means a compound which can reactwith a residue at the catalytically active site of an enzyme toinactivate the enzyme by binding to the site and preventing catalyticactivity.

The term “inactivated” as used herein means that the catalyticallyactive site of an enzyme is covalently coupled to an inhibitor.

The term “reactivation” as used herein refers to the process in whichthe covalent acyl bond is cleaved by hydrolysis, or by a source ofenergy, and enzyme activity is restored.

The term “cinnamic acid” is the common or trivial name for3-phenyl-2-propenoic acid. Z-CINN-X₁-A molecules include derivatives ofcinnamic acid.

IUPAC names are given for synthesized compounds.

For purposes of the present invention “reactive group”, particularly asused to describe the variable B shall be understood to include thosemoieties capable of facilitating conjugation of L and one or morebiologically active moieties or polymers, including but not limited tothe free electron pair (double bond) of a maleimidyl residue.

For purposes of the present invention, the terms “alkyl”, “alkenyl” and“alkynyl” shall be understood to include, e.g., straight, branched,substituted, cyclo-derivatives thereof, including alkoxy or substitutedcycloalkyls, etc. “Substituted” shall be understood to include halo-,alkoxy-, nitro-, etc.

For purposes of the present invention, “molecule” and “biologicallyactive molecule” shall be understood to embrace not only organic orsmall molecules but also proteins, peptides and the like.

For purposes of the present invention, “halogen” shall be understood toinclude chlorine, fluorine, bromine, etc.

II. Z-CINN Derivatives

A. Z-CINN

Various compositions can be formed using Z-CINN as a core molecule. Avariety of functional groups can be introduced into Z-CINN to modify itschemical as well as physical properties. These compositions can alsohave different reactivities toward a nucleophile or light. Somepreferred inhibited compositions are discussed in detail as follows.

The Z-CINN core molecule corresponds to Formula (II)

wherein all variables are as set forth in Formula (I) above.

In one preferred embodiment for Formulas (I) and (II), Z is NR₇R₈.Further, R₁, R₂, R₃, R₄, R₅, and R₆ are H or lower alkyl groups such asCH₃ or CH₂CH₃. In another preferred embodiment for Formulas (I) and(II), R₇ is CH₃CH₂ and R₈ is —(CR₉R₁₀)_(n)—NR₂₂—R₁₁, and R₉ and R₁₀ areH; wherein n is 2; R₂₂ is H or CH₃; and X₁ is O, S or N. In thoseaspects of the invention where J is O and Z is NR₇R₈, some preferred R₈groups include:

-   -   1. (CH₂)_(n),—NH₂ and salts thereof, e.g. —NH₃ ³⁰:Cl⁻ or Br⁻ or        HSO₄ ⁻, etc.    -   2. (CH₂)_(n)—NH—C(O)—H    -   3. (CH₂)_(n)—NH—C(O)—CH₃    -   4. (CH₂)_(n)—NH—C(O)—O-t-butyl    -   5. (CH₂)_(n)—NH—C(O)—O-benzyl    -   6. (CH₂)_(n)—OH    -   7. (CH₂)_(n)—SH    -   8. (CH₂)_(n)—O—C(O)—R₂₁; wherein R₂₁ is CH₃, C₁₋₆ alkyl or        phenyl; wherein each of the above n is an integer of from 2 to        about 10, preferably 2-4.

In still a further aspect of the invention, Z is substituted piperazinethereby providing compositions of the Formula (Ia):

wherein all the variables are as previously defined, with regard toFormula (I).

The cinnamic acid backbone enzyme inhibitors can be synthesized usingstandard techniques and available reagents by those skilled in the art.For example, as FIG. 1 demonstrates, an amine such as2-ethylaminoethylamine (1) can react with an anhydride (2) to generateamide (3). 3 condenses with 1,3-cyclohexanedione (4) followed bydehydration to generate 3-amino-2-cyclohexen-1-one (5). Dehydrogenationof 5 in the presence of a catalyst generates a 3-hydroxyphenyl amine(6). Hydrolysis removes a blocking group to obtain 7, followed byN-acylation to obtain 8. Reaction of 8 with POCl₃/DMF generates analdehyde (9). Reaction of 9 with an appropriate Wittig reagent,carbethoxyethylidene triphenylphosphorane (10), generates 11, where thealdehyde group is converted to a 2-propenoic acid ethyl ester. TheN-acyl group is exchanged to obtain 12, which is hydrolyzed in LiOH toobtain 13 (tBOC-N-CINN). The compounds of Formula (Ia) can be similarlyprepared using piperazine in place of the amine (1).

As FIG. 2 further demonstrates, 13 can be reacted in the presence of analcohol such as 4-aminoiminophenol (14), dicyclohexylcarbodiimide, anddimethylamninopyridine to form an ester (15). The N-protective group canbe hydrolyzed in the presence of HCl to generate a substituted cinnamateester (enzyme inhibitor) containing a free, protonated amino group (16).As FIG. 3 demonstrates, 16 further reacts with a monomeric SS-PEG (17)to generate PEG-derivatized inhibitor (18).

FIG. 4 further illustrates the reaction of 1 with various enzymescontaining active site serines and affinity for 4-aminoiminophenol toobtain inhibited enzyme compositions.

In another embodiment, other alcohols, thiols, or amino compounds, suchas amino acids, amino acid derivatives, peptides, or other compounds areused in place of 14 to form additional-X₁A₁ moieties. For example, anamino acid derivative or peptide derivative is coupled to the N-CINNbackbone (13) via the DCI/DMAP chemistry shown in FIG. 2. Carboxylgroups on a peptide or amino acid would be protected with acceptableblocking groups prior to reaction with 13. The amino group would thenreact with 13, DCI, and DMAP to give an amide bond, and the blockinggroups would be removed. Similar procedures would be followed foralcohols or thiols.

FIG. 5 is the general scheme for preparation of a maleimido derivativeof N-CINN-AP (20), by reaction of (16) with a heterobifunctional reagentcontaining N-hydroxysuccinimide and maleimide. An amino group of 16reacts with a reagent such as SMPH(succinimidyl-6-[β3-maleimidopropionamido) hexanoate; Pierce); themaleimide group at the other end is available for further reactions. Anumber of similar reagents are available, for example bifunctional NHS,bifunctional PEGs, or similar compounds. Those skilled in the art willbe able to choose appropriate linking reagents to couple the Z-CINNbackbone to carrier groups without undue experimentation. For purposesof illustration and not limitation, a non-limiting list of linkers whichcan be employed include succinimides, maleinides, imidoesters,2-iminothiolane, hydrazides, maleic anhydride, azides, citraconicanhydride, glutaraldehyde, and the like, which facilitate attachment ofany desired B group to the Z-CINN core.

FIG. 6 shows that Mal-L-N-CINN-AP (20) can be reacted with a SH-modifiedantibody to form an antibody-L-N-CINN-AP (21). 21 can be reacted withfree enzyme to form antibody-L-N-CINN-enzymne (22), where the enzyme isinhibited. 22 can be treated with light or other energy source toregenerate free enzyme. SH-modified antibody is prepared at˜pH 7 usingcommon reagents and the Mal-L-N-CINN-AP is added at˜pH 7 in slightexcess. Free reagents are separated by ultrafiltration or size exclusionchromatography. In other embodiments, moieties other than SH-modifiedantibodies are used following the schematic set forth in FIG. 6, withchanges in the reactive linking group as required.

B. Inhibition of Enzyme Activity

As defined by Formula I, X₁A may include an active moiety. In thatformula, the active moiety is referred to as the “second” so as todistinguish it from the first active moiety which is part of B and isdiscussed in more detail below. Thus, X₁A₂ can be a biologically activemoiety such as an enzyme which has a serine or cysteine residue at thecatalytically active site, such as a number of proteases or esterases.The enzyme H—X₁A₂ can be specifically reacted with Z-CINN-X₁-A₁, asubstrate or substrate analog for the enzymne. A transition state forms,where the enzyme displaces the leaving group in the acyl bond, and aninhibited acyl-enzyme is formed between the Z-CINN and the enzyme, andis designated herein as Z-CINN X₁-A₂. The inhibited enzyme can bereactivated and recovers its activity when the acyl bond is cleaved byhydrolysis or photolysis.

Exemplary enzymes (H—X₁-A₂) are serine proteinases (proteases) such aschymotrypsin, trypsin, acrosin, clotting factors such as thrombin, VIIa,IXa, Xa, XIa or XIIa, cathepsin G, fibrinolytic pathway proteins such asplasmin, tissue plasminogen activator, urokinase, streptokinase, andcomplement proteins such as C3/C5 convertase, complement Factor I, andcomplement Factor D.

Other exemplary enzymes include cysteine proteinases such as cathepsinB, papain, and bromelain, as well as lipases, and esterases such asacetylcholinesterase.

Preferred serine proteinases include chymotrypsin, trypsin, thrombin,plasmin, acrosin, coagulation factors IXa, Xa, XIa, and XIIa,plasminogen activator, plasma kallikrein, tissue kallikrein, urokinase,plasmin, pancreatic elastase, and leukocyte elastase. Inhibitors arepreferably target specific. For example, imidazole derivatives are knownas good substrates for α-chymotrypsin. F. Marlkwardt et al., Phanazie29:333 (1974); G. Wagner et al., Pharmazie 28:293 (1973); J.Sturzebecher et al., Acta Biol, Med. Germ. 35:1665 (1976); P. Walsmrann,Folia Haematol., Leipzig 109:75 (1982); V. Valenty et al., Biochem.Biophys. Res. Comm. 88:1375 (1979); and F. Markwardt et al., Acta Biol.Med. Germ 28:19 (1972)) describe compounds that lead to stablecarboxylate esters of the enzyme active site serine. Other inhibitorsthat have been studied include compounds that react with the enzyme togenerate stable sulfonate or phosphate esters (See R. Laura et at.,Biochemistry19:4859 (1980); S. Wong and E. Shaw, Arch. Biochiem.Biophys. 161:536 (1974)). Peptide derivatives of phosphonates have alsobeen used (J. Oleksyszyn and JC Powers, Biochemistry 15: 485 (1991)).

Alternatively, A can be a moiety designated herein as A₁. Preferred X₁A₁groups include

in which R₁₂ and R₁₃ are electron donating or electron withdrawinggroups and W is N or CH, or X₁A₁ is an amino acid or amino acidderivative, peptide or peptide derivative, or other substrate orsubstrate analog of enzymes such as serine proteases, cysteineproteases, or esterases containing an active-site serine or cysteinethat can be cleaved by the enzyme, leading to binding of the enzyme tothe cinnamoyl moiety as an acyl-enzyme.

Z-CINN-X₁A₁ represent inhibitors of the enzymes discussed above. Of thepreferred inhibitors, those in which p-nitrophenyl is the leaving group(X₁A₁) are preferred for use in making acyl-enzymes with enzymes such aschymotrypsin. Those inhibitors in which either 4-aminoiminophenyl or4-guanidinophenyl are the leaving groups are preferred for use in makingacyl-enzymes with coagulation enzymes, such as thrombin, plasmin,coagulation Factor IXa, Xa, XIa, XIIa, or with tissue plasminogenactivator, urokinase, and trypsin. Those in which amino acids or aminoacid derivatives, peptides or peptide derivatives, or other substratesor substrate analogs are the leaving groups will react with a variety ofenzymes containing serine or cysteine at the active site, including, forexample, proteases, lipase, esterases, etc.

C. Derivatives of Z-CINN Backbones

A number of different molecules can be attached to the Z-CINN backboneof the present invention. See Formula (III) below.

wherein all variables are as previously defined.

In one aspect, moieties can be attached to increase solubility orcirculating half-life. For example, an activated polyethylene glycol(PEG) or polypropylene glycol (PPG) can be attached to the Z-CINN coremolecule. See U.S. Pat. No. 4,179,337 generally and U.S. Pat. No.5,612,460 which describes inter alia poly(ethyleneglycol)-N-succinimidyl carbonate and derivatives thereof. See also,Poly(ethylene glycol) Chemistry and Biological Applications, Harris, etal., 1997 ACS. The contents of each of the foregoing is incorporatedherein by reference. Generally, with reference to FIG. 3, an activatedmPEG (for example SS-mPEGG₅₀₀₀, Shearwater Polymers, Inc.) is reactedwith 16 at ˜pH 7.0 for 60 min. Excess SS-mPEG is quenched withglycylglycine and the PEG-N-CINN-AP (18) is reacted with an activeserine protease at pH 6.5-7.0 for about 12-16 hours at ambienttemperature to give PEG-N-CTNN-acyl-enzyme (19). This complex can beincluded as part of a pharmaceutically acceptable solution andadministered to a patient in need of such therapy. The conjugate isactivated with light to release native enzyme when needed. It will beappreciated by those of ordinary skill that the specific type ofactivated PEG or other polymer employed will be dependent upon theparticular needs of the artisan and the final product desired. It iscontemplated that most commercially available activated polymers areuseable herein without undue experimentation.

More specifically, in order to form the polymer conjugates of theinvention, R₁₁ may comprise a polymer. For example, polymers such aspolyalklyene oxides (PAOs) or similar biologically acceptable polymersare converted into activated forms, as that term is known to those ofordinary skill in the art. Thus, one or both of the terminal polymerhydroxyl end-groups, (i.e. the alpha and omega terminal hydroxyl groups)are converted into the same (homo-) or different (hetero-) reactivefunctional groups that allow covalent conjugation to the Z-CINN as partof R₁₁, and, if desired another active or targeting group. Othersubstantially non-antigenic polymers are similarly “activated” orfunctionalized. As an alternative to PAO-based polymers, othersubstantially non-antigenic or effectively non-antigenic materials suchas collagen, glycosaminoglycans, poly-aspartic acid, poly-L-lysine,poly-lactic acid, copolymers of the foregoing includingpolylactic-polyglycolic acid copolymers, poly-N-vinylpyrrolidone,collagen cross-linked to hydrophilic polymers or any other suitablenon-reactive polymer such as polyethylene alcohols can be used.Specifically preferred polymeric groups are mono or bifunctionallyactivated polyethylene glycol (PEG) based polymers.

Polymeric groups of any molecular weight range are usable. A preferredpolymer molecular weight can range from 2,000 Daltons to 200,000Daltons. A more preferred molecular weight range is between 5,000 to50,000 D. The most preferred polymer molecular weight range is between12,000 and 40,000 D, (number average).

In another embodiment, a first active molecule (B) is attached toL-Z-CINN-X₁A₁. For example, a protein such as a monoclonal antibody(mAb) can be linked to a Z-CINN-X₁A₁. For example, 16 is reacted with abifunctional reagent containing both NHS and Mal (Pierce Chemical Co.)at pH˜7.0 for 1 hour to give 20 (Mal-L-N-CINN-AP). Excess NHS-Malreagent is quenched with glycylglycine. 20 is then reacted withSH-modified antibody at pH 7.0 for 2 hours at ambient temperature togive 21 (mAb-L-N-CINN-AP). 21 is freed of excess reagent 20 byultrafiltration or get filtration chromatography and can then be reactedwith a serine protease at pH 6.5 for about 12 to 16 hours to give 22(mAb-L-N-CINN-acyl-enzyme). 22 (B-L-N-CINN-X₁A₂) can be targeted to asite with the mAb, then active serine protease is freed by lightactivation.

A non-limiting list of reactive group reagents corresponding to L-B ofR₁₁ include maleimides, N-hydroxysuccinimidyl compounds, imidoesters,2-iminothiolane, hydrazides, maleic anhydride, and the like, which areavailable from Pierce Chemical, for example. The foregoing list ismerely made for purposes of illustration. Once the reactive group isattached, the L-Z-CINN-X₁-A₁ can be attached to a desired molecule (B)such as a targeting mAb using standard conjugation techniques. Anon-limiting list of suitable molecules include antibodies, fragmentsthereof, single chain binding antibodies and the like, includingmoieties that are capable of binding/immobilizing on a particle, forexample, Herceptin® (trastuzumab), other monoclonal antibodies, such asthose directed to cell surface antigens, murine monoclonal antibodies,proteins, nucleic acids, lectins, lipids, carbohydrates, PAOs,glycosaminoglycans, poly-aspartic acid, poly-L-lysine,polyvinylpyrrolidone, collagen, peptides, hormones, ligands forreceptors, and the like. Further specific examples are set forth below:Targeting Agents (including but not limited to the following):

-   Any Antibody that targets a cell or tumor cell in some capacity;-   Monoclonal antibodies (mAbs) with origins from mammals including    mice, rats, humans, monkeys, chimeric constructions, etc.;-   Single chain antibodies;    These antibodies can be expressed in bacteria, plants, yeast,    animals, mammal milk (mouse, goat, sheep, pig, cow, etc), and animal    cell cultures including murine, rat, human, hamster, etc.;-   Growth factors both natural and recombinant and peptide fractions of    growth factors that bind to receptors on the cell surface (EGF,    VEGF, FGF, ILGF-I, ILGF-II, TGF)-   Interferons both natural or recombinant and peptide fractions of    interferons that bind to receptors on the cell surface (IFN-α, β,    and γ) and interferon agonists;-   Cytokines, either natural or recombinant, and peptide fractions of    cytokines that bind to receptor cell surfaces (IL-1, IL-2, IL-3,    IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-12, IL15, TNF, etc);-   Any peptide, natural, recombinant, or synthetic that binds to a cell    surface receptor;-   Any hormone either natural or synthetic that binds to a cell surface    receptor (estrogen, pro-estrogen);-   Any metabolite either natural or synthetic that binds to a cell    surface receptor (amino acids, sugars, vitamins, nucleotide,    nucleoside);-   Any lectin either natural or recombinant that binds to the tumor    cell surface;-   Sugar peptides either phosphorylated or non-phosphorylated (MDP,    MTP, DDP, DTP) or sugar-peptide-lipid targeting agent    (monophosphoryl Lipid A, diphosphoryl-Lipid A, DPG-DDP, DPG-DTP,    etc.);-   Polyethylene glycol polymers and derivatives (2,000->200,000    Daltons);-   Poly-aspartic or glutamic acids or poly-lysine amino acid polymers    or mixtures of these or other amino acids;-   Inhibitors peptides or chemical; covalent binding or non-covalent    binding) of cell surface enzymes (matrix metalloproteinases    inhibitors, tyrosine kinase inhibitors, serine protease inhibitors,    casein kinase inhibitors, plasminogen activator inhibitors); and-   glycoseaminoglycans and dextrans.    As will be apparent to those skilled in the art, those targeting    agents not specifically mentioned, but falling within the above    categories, are also within the scope of the present invention.

Thus, in one aspect of the invention, preferred R₁₁ substituents areproteins such as antibodies to biological materials, which can be usedto assist in the localizing of potential enzyme activity prior torelease by light or other energy source.

D. Carriers and Supports to Immobilize The Z-CINN Derivatives.

Compounds of Formula (I) including Z-CINN core molecule conjugates ofinhibited enzymes can be immobilized on a support made of any of avariety of organic or inorganic materials, including synthetic polymers,natural polymers and biosynthetic polymers. These can be in the form ofcolumns, films, membranes, beads, particles, microparticles, andfilters.

Immobilization is generally achieved by formation of a covalent, ionic,or affinty bond between a side chain of the core inhibitor molecule (forexample 16, N-CINN-AP) and the support material. For example, a supportmaterial containing an aldehyde group or reactive acid group could beused. In one example, agarose beads containing free aldehyde groups(Aldehyde-Agarose; Sigma) are added to 16 at pH˜7.0 to formBead-N-CINN-AP. Excess reagents are washed away and the immobilizedinhibitor can then be reacted with a serine protease at pH 6.5 for 12-16hours at ambient temperature to obtain an immobilized acyl-enzyme, whichcan be reactivated to give serine protease with light. In anotherexample, agarose beads containing N-hydroxysuccinimidyl groups (Sigma)are added to 16 and processed as above. In another example, utilizingaffinity binding, Protein A-Agarose (Sigma or Oncogene) is added to 22at pH 7, mixed by gentle rocking for 90 min, and separated bycentrifiugation. The beads are washed several times to remove unboundmaterial and then can be used to bind enzyme as above, and releaseenzyme activity after exposure to light. These types of binding arewithin the knowledge of one skilled in the art and can be readilyachieved using standard techniques available in the art.

III. Cleavage of Acyl Bond

The covalent acyl bond between the cinnamate core molecule and thetherapeutic moiety designated in Formula (I) as “X₁A₂” can be cleaved byhydrolysis, naturally occurring in solution or, at a controllable rate,following energy input. A variety of energy forms, including light(ultraviolet, visible, or infrared), ultrasound, microwave radiation,radio energy and radioactive decay can be used. Light is most preferred.Light is readily and selectively controllable by variation of thewavelength, duration or energy of the radiation. The preferred energysource is light having a wavelength in the range from 300 to 420 nm,most preferably between 350 nm and 400 nm.

The acyl bond between the enzyme and the enzyme inhibitor can besusceptible to hydrolysis at neutral pH in the dark. The rate ofhydrolysis can be modulated by variation of pH. Its half-life can varyfrom seconds up to several days, The substituted amino group on thecinnamate ring confers stability to the Z-CINN-acyl-enzyme bond thatlasts preferably >24 hr at pH 7.0. This is in contrast to theacyl-enzyme bond reported by King (U.S. Pat. No. 5,770,699) whosehydrolysis rate is designed to be 20-90 minutes. This stability makesthe cinnamate based inhibitors useful for therapeutic applications wherethe inhibited enzyme needs to be kept inhibited in an aqueous systemuntil activation with light. The Z-CINN-acyl-enzyme can therefore alsoserve as a prodrug carrier if desired which releases the enzyme in vivowithout light activation.

Reactivation of the Z-CINN-X₁-A₂ enzyme is accelerated by light energy.Energy input induces a conformational change in the Z-CINN portionleading to hydrolysis of the acyl-enzyme bond and release of activeenzyme. The preferred energy sources have a wavelength in the range from300 to 420 nm for reactivation times ranging from seconds to minutes.The extent of reactivation depends on such factors as the nature of theinactivated enzyme, the environment, and the type and intensity of theenergy source. For example, inactivated thrombin can be readilyreactivated by application of white light in a period of about twentyseconds.

The energy source can be introduced via means such as an externalvisible light source like a focused lamp or laser. A fiber opticcatheter that emits light from its tip can deliver light to the target.In other embodiments, the energy source can be introduced in the form ofmicrowave irradiation, radio wave irradiation, ultrasound, radiation ofradioactive materials, ultraviolet irradiation, or infrared irradiation.

IV. Methods of Use/Treatment

The compounds disclosed herein have many applications. One generalapplication of pharmaceutical importance uses the bifunctional aspect ofthe cinnamate backbone to target an enzyme to some specific structure inthe body. These structures could be on tumor cells or extracellular,such as a blood clot. Another use is to inhibit an enzyme, which is notneeded or desired initially in an application, and reactivate it laterin the procedure. The inactivated enzyme can be reactivated by applyinglight, ultrasound, or other energy source to cleave the acyl bondbetween the inhibited enzyme and the inhibitor. Another application isto increase the stability or circulatory lifetime of the inhibitedenzyme in solution. The amount of the compound administered to a mammalin need of such treatment will of course depend upon the needs of thepatient and the enzyme or therapeutic agent administered. It iscontemplated that the amount administered will be based on known amountsfor the unconjugated enzyme and will be sufficient to successfully treatthe mammal.

The compounds of the present invention are thus administered as part ofpharmaceutically acceptable formulations e.g. as part of parenteral,i.e. intravenous or oral dosage forms as such are known to those ofordinary skill in the art.

The compounds can be used for purification, particularly those that areimmobilized and bind selectively with a target molecule under a givenset of conditions, by allowing solutions of the targeted enzyme toequilibrate with inhibitor, then washing away solution before releasingfree enzyme following energy input. For example, a compound containing4-aminoiminophenol at the “A” position could be immobilized and used toisolate thrombin from solution.

The compounds can be used in diagnostic assays. The diagnostic assay maybe manual or automated, useful either in laboratories or in the form ofa kit.

V. Methods of Synthesis

In another aspect of the invention there are provided methods ofpreparing the conjugates described herein. Some preferred methodsinclude reacting a compound of Formula (IV):

wherein all variables are as defined with respect to Formula (I) and R′₇is selected from among H, CH₃ and C₂-C₁₀ alkyls;

-   -   with a compound of the Formula (V)        L₁−B₁  (V)    -   wherein L₁ is a moiety containing a functional group capable of        reacting with the NH₂ of Formula IV);    -   and B₁ is polymer, a biologically active material such as an mAb        or fragment, etc. polymeric support or the like;        under conditions sufficient to form a conjugate corresponding to        Formula (VI)

wherein L₁′ represents the residue of L₁ after the reaction of (IV) with(V). For purposes of the present invention, “conditions sufficient”shall be understood to include not only those conditions set forth inthe Examples below but also those conditions, e.g. temperatures,suitable reagents, solvents, etc. associated with achieving the desiredfinal product.

Some particularly preferred compounds of the present invention are setforth in the following examples and figures. See, for example, compounds16, 18, 20 and 21.

The compounds and methods of use thereof disclosed herein can be furtherunderstood by the following non-limiting examples.

VI. EXAMPLES

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described below are offeredby way of example only, and the invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled.

Example 1 Synthesis of3-[2-hydroxy-4-(ethyl)(2-butyloxycarbamidoethyl)-amino]phenyl-2-methyl-2-propenoicacid (13)

Step 1. Synthesis of (2-ethylaminoethylcarbamic acid, 1,1-dimethylethylester) (3)

Refer to FIG. 1. A 25g (0.28 mol) quantity of N-ethylethylenediamine (1)was placed in a dried 500 ml rb flask. THF, 250 ml, previously dried bydistillation from CaH, was added via cannula. The flask was kept underargon and cooled in an ice bath for 20 minutes. A dried addition funnelwas charged with 50 ml THF to which was added 29.3 ml (0.13 mol) ofdi-tert-butyl dicarbonate (2). This mixture was added slowly dropwise tothe stirred amine. After addition was complete, the reaction was removedfrom the ice bath and allowed to stir and reach ambient temperatureovernight. The volatiles were removed by rotary evaporation. SaturatedNaCl (50 ml) was added, and the result extracted with 4×100 ml ethylacetate. The combined organic fractions were dried over Na₂SO₄overnight. The drying agent was removed by filtration and the volatilesremoved by rotary evaporation to yield 2-ethylaminoethylcarbamic acid,1,1-dimethylethyl ester (3; 27.55 g, 0.15 mol). The material was usedwithout further purification.

Step 2. Synthesis of(2-(ethyl)(3-cyclohex-2-ene-1-one)aminoethylcarbamic acid,1,1-dimethylethyl ester) (5)

A 200 ml quantity of benzene was added to 27.5 g (0.15 mol)2-ethylaminoethylcarbamic acid, 1,1-dimethylethyl ester (3) in a 500 mlrb flask. The flask was equipped with a Dean-Stark trap and condenser.The mixture was brought to reflux under argon and 18 g (1.1 eq.) of1,3-cyclohexanedione (4) was added all at once. The benzene/water layerwas removed from the trap, about 75 ml overall. The trap was rinsed withacetone, dried briefly and quickly replaced and filled with freshlyactivated 4A molecular sieves and the reaction allowed to proceedovernight. The reaction was cooled to ambient temperature under argon.The volatiles were removed by rotary evaporation to yield a red-orangeoil, which may solidify on standing. The material(2-(ethyl)(3-cyclohex-2-ene-1-one)aminoethylcarbamic acid,1,1-dimethylethyl ester; 5) was used without further purification.

Step 3. Synthesis of (2-(ethyl)(2-hydroxyphenyl)aminoethylcarbamic acid,1,1-dimethylethyl ester) (6)

To 0.15 mol crude 2-(ethyl)(3-cyclohex-2-ene-1-one)aminoethylcarbamicacid, 1,1-dimethylethyl ester (5) was added 300 ml CH₃CN, freshlydistilled from CaH. The mixture was brought to reflux under argon and1.1 eq. mercuric acetate, Hg(AcO)₂, was added in one portion. Thesuspension was stirred at reflux overnight under argon. Reaction wasaccompanied by a color change to a claret solution, a violet precipitateand elemental Hg. The reaction was allowed to cool to ambienttemperature under argon and was then transferred to centrifuge tubes andcentrifuged at 3000 rpm for 10 minutes. The supernatant solutions wereremoved and combined. Additional CH₃CN was added to the tubes; the solidprecipitate was resuspended and centrifuged as above. The supernatantswere pooled with those previously obtained. The volatiles were removedby rotary evaporation. The dark purple isolate was transferred to aseparatory funnel with a minimal volume of chloroform and the resultingsolution was washed with 5% NaHCO₃ until the pH of the aqueous layer wasabout 7. The organic layer was removed and two additional extractionswere performed with chloroform, followed by three extractions with ethylacetate. The organic fractions were combined, dried over Na₂SO₄ andfreed of volatiles by rotary evaporation. The crude material obtained inthis way can be stored under argon at −80° C. for up to several days. A680 g quantity of silica was mixed with 134 g decolorizing charcoal,slurried with hexane and poured into a 100 mm glass column with about190 mm of packing. The crude isolate was loaded in a minimum volume ofchloroform (3×50 ml). The elution was performed at 7-10 psi with ethylacetate/hexane: 1.5 L, 20%; 1.5 L, 30%; 1.5 L, 40%; 1 L, 50%. Fractionswere pooled and volatiles removed by rotary evaporation to leave aslightly yellowish oil. The material(2-(ethyl)(2-hydroxyphenyl)aminoethylcarbamic acid, 1,1-dimethylethylester; 6) exhibits behavior consistent with sensitivity to air and lightat this point and must be handled accordingly. It was successfullystored at −20° C. under argon for a period of one month. Yield: 8.78 g(21% for two steps).

Step 4. Synthesis of (3-(ethyl)(2-acetylaminoethyl)aminophenol)(8)

Degassed ethyl acetate (75 ml) was added to 5.5 g (19.6 mmol)2-(ethyl)(2-hydroxyphenyl)aminoethylcarbamic acid, 1,1-dimethylethylester (6). To this was added 50 ml of 3N HCl and the reaction wasstirred at ambient temperature overnight. TLC analysis was done using30% EtOAc/hexanes. The reaction mixture was tranferred to a separatoryfunnel and extracted with 2×40 ml 1N HCl. The pooled aqueous layers wereplaced on the rotary evaporator and the volatiles removed. Water wasadded (about 50 ml) and removed by rotary evaporation, 2 cycles. Theresulting oil was frozen in dry ice and placed under vacuum, thenallowed to warm naturally to remove all volatiles. The resulting crispymaterial (7) was used without further purification, and kept free of airand moisture. Dry pyridine (75 ml) was added to the isolate by cannula.The flask was swirled to dissolve the material. Acetic anhydride (1.2eq.) was added via syringe and the reaction was stirred under argon atambient temperature. TLC analysis was performed with 10%methanol/chloroform. At the completion of the reaction, the pyridine wasremoved by rotary evaporation and the residue treated with 100 ml of 10%NaOH by stirring overnight under argon. The reaction was adjusted to pH9 with 1N HCl and extracted with 4×75 ml ethyl acetate. The combinedorganic layers were washed with water, then brine (once each), pre-driedwith Na₂SO₄, then dried overnight with MgSO₄. The solution was collectedby filtration and the volatiles were removed by rotary evaporation. Thematerial was purified by flash chromatography on silica: loaded with 5%methanol/chloroform; eluted with 150 ml each of 3.3, 6.6, and 9.9%solvent as above to give 3-(ethyl)(2-acetylaminoethyl)aminophenol (8).Yield was 4.2 g (96%).

Step 5. Synthesis of(2-hydroxy4-(ethyl)(2-acetylaminoethyl)aminobenzaldehyde) (9)

A scrupulously dry flask was charged with anhydrous DMF (15 ml) andcooled in ice under argon. Phosphorus oxychloride, POCl₃, 1.5 ml (1.15eq) was added slowly dropwise to the stirred solution. After theaddition was complete, the flask was removed from the ice bath andallowed to reach ambient temperature. This was accompanied by a colorchange to yellow. The flask was heated in a 40° C. bath for one hour. Itwas removed from the heat, and once at ambient temperature, a DMFsolution of 3-(ethyl)(2-acetylaminoethyl)aminophenol (8; 3.2 g, 14 mmol)was added via addition funnel. After the addition was complete, theflask was returned to the 40° C. bath and stirred overnight. Thereaction was cooled to 0° C. and 25 ml 10% NaOH was added, the mixturewas allowed to warm to ambient temperature and was then placed in a 40°C. bath. Additional NaOH was added to achieve pH >10. Water (50 ml) wasadded. After 15 minutes the flask was removed from the bath and the pHadjusted to 5-5.5 with 1N HCl. The mixture was transferred to aseparatory funnel and extracted with 4×100 ml ethyl acetate. The pooledorganic fractions were washed with water (1×75 ml), predried withNa₂SO₄, and then dried with MgSO₄ overnight. The solution was collectedby filtration and the volatiles removed by rotary evaporation to yieldthe crude product (2.9 g). The material was purified by flashchromatography on silica, 45×150 mm column, eluting with 200 ml each of2.5%, 5%, and 10% methanol/chloroform purified yieldof(2-hydroxy-4-(ethyl)(2-acetylaminoethyl)aminobenzaldehyde) (9) was1.52 g (45%).

Step 6. Synthesis of(3-[2-hydroxy-4-(ethyl)(2-acetylaminoethyl)amino]phenyl-2-methylpropenoicacid, ethyl ester) (11)

2-hydroxy-4-(ethyl)(2-acetylaminoethyl)aminobenzaldehyde (9;1.58 g, 6.3mmol) was placed in a flask with degassed benzene (50 ml).Carbethoxy(ethylidene)triphenylphosphorane (10;1.05 eq.) was added andthe reaction stirred overnight under argon. The reaction was freed ofvolatiles by rotary evaporation, then purified by flash chromatography(silica, 95 mm×45 mm), eluting with 250 ml 2.5%; 150 ml 5%; and 150 ml10%; all methanol/chloroform to give3-[2-hydroxy-4-(ethyl)(2-acetylaminoethyl)amino]phenyl-2-methylpropenoicacid, ethyl ester (11). The yield was quantitative.

Step 7. Synthesis of(3-[2-hydroxy4-(ethyl)(2-tert-butyloxycarbamidoethyl)-amino]phenyl-2-methylpropenoicacid, ethyl ester) (12)

3-[2-hydroxy-4-(ethyl)(2-acetylaminoethyl)amino]phenyl-2-methylpropenoicacid, ethyl ester (11; 2.0 g, 6 mmol) was dissolved in anhydrous THF (30ml). Di-t-butyldicarbonate (2; 3 eq) and dimethylaminopyridine (0.1 eq)were added and the reaction was heated to 75° C. TLC analysis (silica,5% methanol/chloroform) was used to gauge reaction completion; after 3.5hours the reaction was determined to be finished. The solution wasallowed to cool to ambient temperature, the volume of the reaction wasdoubled with anhydrous methanol, and hydrazine (3 eq.) was addeddropwise via syringe. The reaction was allowed to stir under argonovernight. The solution was poured into 100 ml CH₂Cl₂ in a separatoryfunnel, and water (100 ml) was added. The mixture was shaken and allowedto separate; the water layer was adjusted to pH 4.5 by judiciousaddition of 1N HCl, shaking well after each addition. The aqueous layerwas cloudy but colorless. After thorough washing, the layers wereseparated and the organic layer washed with aqueous NaHCO₃ at pH7.8-8.0. The organic layer was dried by stirring overnight over MgSO₄.The solution was collected by filtration and the volatiles were removedby rotary evaporation. The material was purified by flash chromatographyon silica, using 400 ml 15% and 300 ml 30% ethyl acetate/hexane to give3-[2-hydroxy-4-(ethyl)(2-tert-butyloxycarbamidoethyl)amino]phenyl-2-methylpropenoicacid, ethyl ester (12). The yield was 1.7 g (72%).

Step 8. Synthesis of3-[2-hydroxy-4-(ethyl)(2-tert-butyoxycarbamnidoethyl)-amino]phenyl-2-methylpropenoicacid (13)

3-[2-hydroxy-4-(ethyl)(2-tert-butyloxycarbamidoethyl)amino]phenyl-2-methylpropenoicacid, ethyl ester (12;759 mg, 1.9 mmol) was taken up in ethanol (10 ml).A 5 ml quantity of 10% w/v LiOH was added and the reaction was heated to40° C. under argon. The reaction was complete after 6 hours, determinedby TLC. The mixture was allowed to cool to ambient temperature, the pHadjusted to 5-6 with 1N HCl, and extracted into ether (4×50 ml); thecombined organic layers were washed with saturated NH₄ ⁺Cl⁻ and driedover CaSO₄ overnight. The solution was collected by filtration and thevolatiles were removed by rotary evaporation to give3-[2-hydroxy-4-(ethyl)(2-tert-butyloxycarbamidoethyl)amino]phenyl-2-methylpropenoicacid (13). The material was used without further purification. Yield was509 mg (74%).

Example 2 Preparation of3-[2-hydroxy-4-(ethyl)aminoethyl)amino]phenyl-2-methylpropenoic acid,4-aminoiminophenyl ester, HCl salt (16; N-CINN-AP)

Step 1. Synthesis of3-[2-hydroxy-4-(ethyl)(2-tert-butyloxycarbamidoethyl)amino]-phenyl-2-methylpropenoicacid, 4-aminoiminophenyl ester, HCl salt (15; tBOC-N-CINN-AP)

Refer to FIG. 2.3-[2-hydroxy-4-(ethyl)(2-tert-butyloxycarbamidoethyl)-amino]phenyl-2-methylpropenoicacid (13) (509 mg; 1.4 mmol) was dissolved in anhydrous pyridine.Dicyclohexylcarbodiimide (1.2 eq.), 4-aminoiminomethylphenolhydrochloride (14; 1.1 eq.) and dimethylaminopyridine (0.05 eq.) wereadded and stirred at ambient temperature under argon for 12-16 hr. Thereaction mixture was passed through a medium porosity sintered glassfilter, and freed of volatiles by rotary evaporation. The isolate wastaken up in 5% methanol/chloroform, filtered, and freed of volatiles asabove. The crude material, 15, was purified by flash chromatography onsilica, loading with 3 ml 5% and eluting with 100 ml 5%; 75 ml 10%; 75ml 15%; and 200 ml 20%; all methanol/chloroform. Yield was 448 mg (66%).

Step 2. Synthesis of3-[2-hydroxy-4-(ethyl)aminoethyl)amino]phenyl-2-methylpropenoic acid,4-aminoiminophenyl ester, HCl salt (16; N-CINN-AP)

3-[2-hydroxy-4-(ethyl)(2-tert-butyloxycarbamidoethyl)amino]phenyl-2-methylpropenoicacid, 4-aminoiminophenyl ester, HCl salt (15; 12 mg, 25 μmmol) wassuspended in 1.2 ml of 3N HCl and mixed by inversion for 3 hours atambient temperature to release the t-BOC group and generate3-[2-hydroxy-4-(ethyl) aminoethyl)amino]phenyl-2-methylpropenoic acid,4-aminoiminophenyl ester, HCl salt (16; N-CINN-AP). After 3 hours, thereaction was neutralized with 3N NaOH to give a final pH of 7.0. Thismaterial was used immediately and was not characterized further exceptthat it was now soluble in aqueous solution.

Example 3 Preparation of mPEG₅₀₀₀-N-CINN-AP (18)

Refer to FIGS. 3 and 4. mPEG₅₀₀₀-SS (2M4R0H01; Shearwater Polymers) wasused as follows.3-[2-hydroxy-4-(ethyl)aminoethyl)amino]phenyl-2-methylpropenoic acid,4-aminoiminophenyl ester, HCl salt (16; 2.5 mg, 6.5 μmol; pH 7.0-7.4) in1 ml of 10 mM sodium phosphate buffer, pH 7.2, was added to drymPEG₅₀₀₀-SS (17; 38.3 mg, 7.5 μmol). After 1 hour at ambienttemperature, the reaction was stopped by the addition of 75 μMglycylglycine in 100 μl of water. This material (mPEG₅₀₀₀-NH-CINN-AP;18)was used within 3 hours for inhibition reactions with serine proteasessuch as thrombin and tPA.

Example 4 Inhibition of Thrombin with R—NH-CINN-AP Derivatives

R is -tBOC, —H₂ ⁺, or mPEG₅₀₀₀. Human thrombin was modified withR—NH-CINN-AP inhibitors as follows: Thrombin (600 units; ˜3.5 nmol) in50 mM sodium citrate buffer, 3 mM CaCl₂, 7% kollidon, pH 7.0, wasreacted with 3.5-35 nmol of the R—N-CINN-AP inhibitor for 12-18 hours atambient temperature in the dark. Thrombin activities were measured underred light (“dark”) and after exposure to 10 seconds of blue light from adental light and were assayed using S-2238 (diaPharma) at 30° C.Percentage inhibition was calculated as 100—dark activity. Dark activitywas calculated as [activity_(dark)÷activity_(control)]×100. Lightreactivation was calculated as activity_(light)÷activty_(control). Thefollowing are typical results.

TABLE 1 Inhibition and Reactivation of R-N-CINN-Acyl-Thrombin^(a)Inhibitor Inhibition after 16 Light Inhibitor Excess Hrs Incubation (%)Reactivation (%) None 0  0 NP CINN-AP 1× 53 NP CINN-AP 5× 99 NP CINN-AP10×  99 60 #15 1× 94 NP #15 5× 98 NP #15 10×  97 72 #16 1× 21 NP #16 5×70 NP #16 10×  96 64 #18 2× 51 NP #18 10×  98 69 ^(a)Thrombin activitywas assayed as described in Chromogenic Substrates (Chromogenix ABInformation Manual) using S-2238 (H-D-Phe-Pip-arg-pNA•2HCl) as thesynthetic substrate. #15 = tBOC-N-CINN-AP #16 = NH₃-CINN-AP #18 =mPEG₅₀₀₀-N-CINN-AP NP = Assay not performed

The R—NH-CINN-AP compounds inhibit thrombin, as does the parent compoundCINN-AP. All of the acyl-thrombins can be reactivated with blue light,indicating that the modifications to create R—NH-CINN-AP do not affectthe ability of the molecule to isomerize and release free enzyme fromthe acyl-enzyme. The NH₃-CINN-AP, which contains a positive charge,appears to be a less effective inhibitor at lower molar ratios. ThemPEG₅₀₀₀-NH-CINN-AP, which contains a 5000 dalton linear mPEG molecule,requires higher concentrations to effect thrombin inhibition. Theseresults show that one can alter other portions of the molecule withoutlosing the effects of a light reversible, covalent inhibitor. Lightreactivation remains essentially the same, indicating that theR—NH-CINN-acyl-thrombin molecule absorbs blue light energy and canisomerize to give the cis configuration and release active thrombin.

Example 5 Inhibition of Tissue Plasminogen Activator (tPA) withR_(z)—NH-CINN-AP Derivatives

R₂ is -tBOC, —-H₂ ⁺, or mPEG₅₀₀₀. The procedures of Examnple 4 wasrepeated except that Human tPA (Activase, Genentech Inc. Lot # E9034A)was modified with R_(z)—NH-CINN-AP inhibitors in place of thrombin. Thereactions were carried out as follows: tPA (0.83 nmol) in 10 mMtris-HCl, 130 mM NaCl, pH 7.4 was reacted with 0.8 3-8.3 nmoles of theR—NH CINN-AP inhibitors for 12-18 hours at ambient temperature in thedark. Activities were calculated as for Example 4, using S-2288(diaPharna). The results are provided below.

TABLE 2 Inhibition and reactivation of R_(z)-N-CINN-tPA^(a) InhibitorInhibition after 16 hrs Light Reactivation Inhibitor Excess incubation(%) (%) None 0  0 NP CINN-AP 5× 95 NP CINN-AP 10×  95 82 #15 5× 95 NP#15 10×  95 87 #16 5× 78 NP #16 10×  88 78 ^(a)tPA activity was assayedas described in Chromogenic Substrates (Chromogenix AB InformationManual) using S-2288 (H-D-Ile-Pro-Arg-pNA•2HCl) as the syntheticsubstrate #15 = tBOC-N-CINN-AP #16 = NH₃-CINN-AP NP = Assay notperformed

The R_(z)—NH-CINN-AP compounds inhibit tPA, as does the parent compoundCINN-AP. All of the acyl-tPAs can be reactivated with blue light,indicating that the 30 modifications to create R—NH-CINN-AP do notaffect the ability of the molecule to isomerize and release free enzymefrom the acyl-enzyme. The NH₃-CNN-AP, which contains a positive charge,appears to be a less effective inhibitor at lower molar ratios. Theseresults show that other portions of the molecule can be altered withoutlosing the effects of a light reversible, covalent inhibitor. Lightreactivation remains essentially the same, indicating that theR—NH-CINN-acyl-tPA molecule absorbs blue light energy and can isomerizeto give the cis configuration and release active tPA.

Example 6 Preparation of N-CINN-AP with Maleimide Linker (20)

N-CINN-AP (16; 10 μmoles, 1 ml) was reacted with 11.4 μmoles of SMPH(succinimidyl-[β-maleimidopropionamido] hexanoate; Pierce #22363) in 0.5ml DMSO and 0.5 ml 200 mM sodium phosphate, pH 7.4 for 2 hours atambient temperature. After 2 hours, the reaction was placed on ice, andunreacted NHS was quenched with 0.05 ml of 100 mM glycylglycine. Thefinal product (20) was used within 2 hours, and was not furthercharacterized.

Example 7 Preparation of Antibody-Linker-N-CINN-AP (21)

A monoclonal antibody, Herceptin® (trastuzumab, Genentech) was preparedat 5 mg/ml in phosphate-NaCl buffer, pH 7. SATA (N-succinimidylS-acetylthioacetate; Pierce) was dissolved in DMSO and a 20-fold molarexcess was added to the protein and reacted for 1 hour. HydroxylamineHCl (Pierce; 0.5 M, neutralized to pH 7.0) was added for 60-90 minutes,and antibody was separated from low molecular weight material bychromatography on D-SALT Polyacrylamide desalting columns (Pierce)equilibrated in phosphate-NaCl buffer. Incorporation of thiols intoantibody (antibody-SH) was determined using Ellman's reagent (Pierce).20, at a 2-fold molar excess to incorporated-SH groups, was added toAntibody-SH for 2 hours at pH 7.4 and ambient temperature to give 21.Antibody-containing fractions (21) were separated from excess 20 on aD-SALT column as above.

Example 8 Preparation of Antibody-Linker-N-CINN-Enzyme (22)

21 was mixed with thrombin (400 U/ml; Enzyme Research Labs) at pH 7.0for 12-16 hours at ambient temperature to form 22, and 22 was separatedfrom free enzyme by binding to Protein A-Agarose beads (Oncogene) whichhad been washed 3 times with phosphate-NaCl buffer. After gentle mixingfor 90 min, the beads were washed by centrifulgation 3 times andsupernatants containing unbound enzyme were removed. Beads weresuspended in assay buffer at pH 8.3 containing 0.5 mM S-2238(Chromogenix), and assayed for 6 min at 30° C. Ten seconds of blue lightfrom a ProLite dental light was then directed to the wells, and assayswere continued for another 6 min. The rate of S-2238 cleavage increased7-8 fold on exposure to light, indicating release of free thrombin intosolution.

Example 9 Preparation of Antibody-PEG-N-CINN-AP (23)

N-CIN-AP (16, 2.4 μmoles, 1.0 ml) in 50 mM sodium phosphate buffer, pH7.0, was reacted with 4.8 μmoles of PEG₃₄₀₀-(SPA)₂ (PEG succinimidylpropionate; Shearwater Polymers, Inc) in 1 ml WFI at ambient temperaturefor 1 hour. The SPA-PEG-N-CINN-AP was immediately reacted with goatanti-rabbit IgG (Calbiochem, 401311) as follows: 10.6 nmoles ofSPA-PEG-N-CINN-AP was reacted with 2.6 moles antibody for 2 hours at pH7.0 at ambient temperature. The reaction mixture was placed on ice andexcess SPA was quenched with 0.05 ml of 100 mM glycylglycine. Theproduct was used within two hours, and not further characterized.

Example 10 Preparation and Assay of Antibody-PEG-N-CINN-Acyl-Thrombin(24)

Antibody-PEG-N-CINN-AP (23) was mixed with thrombin (400 U/ml; EnzymeResearch Labs) at pH 7.0 for 16 hours at ambient temperature. Theantibody-acyl-enzyme complex was then tested for its ability to bind toits antigen and release active enzyme. Rabbit IgG (Calbiochem) was boundto polystyrene plates following standard procedures. The plates werethen blocked with PBS/Tween 20/BSA (Sigmna) for 2 hours, and washedextensively with PBS/Tween 20 to remove excess protein. Goat anti-rabbitantibody-PEG-N-CINN-Acyl-Thrombin (24) was then allowed to bind to therabbit IgG on the plate for 2 hours at ambient temperature. The plateswere then washed 5 times with PBS/Tween 20 to remove any unboundantibody-thrombin or free thrombin. The plates were assayed for thrombinactivity using the S-2238 chromogenic assay described in Example 8. Therate of S-2238 cleavage increased 5 fold after 10 seconds of lightactivation indicating release of free thrombin into solution.

Examples 11-13 Murine nAbs as Targeting Agents

The following examples provide details regarding the preparation ofspecific murine mAb-AZ-CINN conjugates and their utility as targetingagents for therapeutic indications.

Example 11 Lung Cancer

A. Preparation of Antibody-Linker-CINN-Enzyme and Its Use

N-CINN-AP (16,2.4 μmoles, 1.0 ml) in 50 mM sodium phosphate buffer pH7.0 is reacted with 4.8 μmoles of NHS-PEG₃₄₀₀-MAL (Shearwater Polymers,Inc. NHS-Mal-3400) dissolved in DMSO. The reaction mixture is rocked fortwo hours at ambient temperature. The reaction is stopped by theaddition of 100 μl of 100 mM glycylglycine. The Mal-PEG₃₄₀₀-N-CINN-AP isused within two hours and not further characterized.

B. Preparation of Antibody-PEG-N-CINN-Acyl-Thrombin

A murine monoclonal antibody, BR110 (Hellstrom et.al. U.S. Pat. No.5,840,854) is diluted to 5 mg/ml in phosphate-NaCl buffer, pH 7. SATA(N-succinimidyl S-acetylthio-acetate; Pierce) is dissolved in DMSO and a20-fold molar excess is added to the protein and reacted for 1 hour.Hydroxylamine HCl (Sigma; 0.5 M, neutralized to pH 7.0) is added for60-90 minutes, and antibody is separated from low molecular weightmaterial by chromatography on D-SALT Polyacrylamide desalting columns(Pierce) equilibrated in phosphate-NaCl buffer. Incorporation of thiolsinto the murine antibody (antibody-SH) is determined using Elman'sreagent (Pierce). Mal-PEG₃₄₀₀-N-CINN-AP at a 2-fold molar excess toincorporated-SH groups, is added to BR110-mAb-SH for 2 hours at pH 7.4and ambient temperature to give BR110-mAb-S-Mal-PEG₃₄₀₀-N-CINN-AP.Antibody-containing fractions (BR110-mAb-S-Mal-PEG₃₄₀₀-N-CINN-AP) areseparated from excess Mal-PEG₃₄₀₀-N-CINN-AP on a D-SALT column (Pierce)buffered with 50 mM sodium phosphate pH 6.5 and 150 mM NaCl. Theantibody-drug (BR110-mAb-S-Mal-PEG₃₄₀₀-N-CINN-AP) is concentrated to 5mg/ml protein using Centricon YM-30 membranes and sterile filtered (0.22μm filter; Gelman Sciences, Supor Acrodisc 25).BR110-mAB-S-Mal-PEG-N-CINN-AP is mixed with thrombin (400 U/ml; EnzymeResearch Labs) at pH 7.0 for 16 hours at ambient temperature. Theantibody-acyl-enzyme complex is then separated from free thrombin andconcentrated to 5 mg/ml protein using Centricon YM-100 membranes andsterile filtered (0.22 μm filter; Gelman Sciences, Supor Acrodisc 25).

C. Treatment of Human Lung Carcinoma withBR110-mAb-S-Mal-PEG3400-N-CINN-Acyl-Thrombin

MAb BR110 is a murine mAb to a 66-kDa glycoprotein that is found on thecell surface of human lung, colon and breast tumor cells. Human lungcarcinoma cell line H2987 is grown in cell culture as described (U.S.Pat. No. 5,840,854). 1×10⁷ cells/ mouse are implanted s.c. into a nudemice. When the tumors reach 150-200 mm³ in size,BR110-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin is used to treat the tumoras follows; 1.0 mg of BR 110-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin (1mg mAb protein with 27 nmoles thrombin covalently attached) is injectedi.v. via tail vein into mice with the tumor. After 6 hours, mice thatare given the BR110-mAb-S- Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin areanesthetized with a Ketamine/Rompun mixture. A 16G needle is used tocreate a hole in the skin and a 1 mm fiber optic probe is inserted up tothe tumor. The tumor is exposed to white light for 5 minutes with apower of about 0.5 mW/cm². Other treatment groups do not receive lighttreatment. All mice are kept under “red lights” to prevent lightactivation of the drug, for 72 hours post injection, then returned tonominal light/dark cycle. Tumors in groups that receive light treatmentshrink immediately (>50% in 7 days) and are necrotic. Tumors that aretreated with BR110-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin but do notreceive light stimulation shrank at a much slower pace and are smallerthan 100 mm³ at 28 days. Groups that receive antibody and free drug(unlinked) show tumor growth, and by 28 days the mice either die or havetumors >800 mm³.

Example 12 Colon Cancer

A. Preparation of Antibody-Linker-CINN-Acyl-Thrombin

Mal-PEG₃₄₀₀-N-CINN-AP is prepared as described above in Example 11A.

B. Preparation of Antibody-PEG-N-CINN-Acyl-Thrombin

The procedure of Example 11B is repeated except that MAb A7, a murinemAb to a 45-kDa glycoprotein found on human colon cancer cells, is usedin place of the BR110-mAb to produceA7-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin.

C. Treatment of Human Colon Carcinoma withA7-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin

Human colon carcinoma cell line LS-180 is grown in cell culture asdescribed (Kinuya et. al. 2001. J. Nucl. Med. 42:596-600). 1×10⁷cells/mouse are implanted s.c. into nude mice. When the tumors reach150-200 mm³ in size, A7-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin is usedto treat the tumor as follows; 1.0 mg ofA7-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin (1 mg mAb protein with˜27nmoles of thrombin covalently attached) is injected i.v. via tail veininto mice with the tumor. After 6 hours, mice that are given theA7-mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin are anesthetized with aKetamine/Rompun mixture. A 16 G needle is used to create a hole in theskin and a 1 mm fiber optic probe is inserted up to the tumor. The tumoris exposed to white light for 5 minutes with a power of about 0.5mW/cm². Other treatment groups do not receive light treatment. All miceare kept under “red lights” to prevent light activation of the drug, for72 hours post injection, then returned to normal light/dark cycle.Tumors in groups that receive light treatment shrink immediately (>50%in 7 days) and are necrotic. Tumors that are treated withA7-mAb-S-Mal-PEG₃₄₀₀-N-CN-Acyl-Thrombin but do not receive lightstimulation shrink at a much slower pace and are smaller than 100 mm³ at28 days. Groups that receive antibody and free drug (unlinked) showtumor growth, and by 28 days the mice either die or have tumors>800 mm³.

Example 13 Breast Cancer

A. Preparation of Antibody-Linker-CINN-Acyl-Thrombin

Mal-PEG₃₄₀₀-N-CINN-AP is prepared as described above.

B. Preparation of Antibody-PEG-N-CINN-AcyI-Thrombin

The procedure of Example 11B is repeated except that MAb NR-LU-10, amurine mAb to a pancarcinoma glycoprotein found on human breast cancercells, is used in place of the BR110-mAb to produce NR-LU-10S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin.

C. Treatment of Human Breast Carcinoma with NR-LU-10mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acl-Thrombin

Human breast cancer xenografts are prepared as described (Burak et.al.1998. Nucl. Med. Biol. 25:633-637) Xenografts are implanted s.c. intonude mice. When the tumor reaches 150-200 mm³ in size, NR-LU-10mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin is used to treat the tumor asfollows; 1.0 mg of NR-LU-10 mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin (1 mgmAb protein with˜27 nmoles of thrombin covalently attached) is injectedi.v. via tail vein into mice with the tumor. After 6 hours, mice thatare given the NR-LU-10 mAb-S- Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin areanesthetized with a Ketatine/Rompun mixture. A 16 G needle is used tocreate a hole in the skin and a 1 mm fiber optic probe is inserted up tothe tumor. The tumor is exposed to white light for 5 minutes with apower of about 0.5 mW/cm². Other treatment groups do not receive lighttreatment. All mice are kept under “red lights” to prevent lightactivation of the drug, for 72 hours post injection, then returned tonormal light/dark cycle. Tumors in groups that receive light treatmentshrink immediately (>50% in 7 days) and are necrotic. Tumors that aretreated with NR-LU-10 mAb-S-Mal-PEG₃₄₀₀-N-CINN-Acyl-Thrombin but do notreceive light stimulation shrink at a much slower pace and are smallerthan 100 mm³ at 28 days. Groups that receive antibody and free drug(unlinked) show tumor growth, and by 28 days the mice either die or havetumors>800 mm³.

The various publications, patents, patent applications and publishedapplications mentioned in this application are hereby incorporated byreference herein.

1. A compound of the formula:

wherein: R₁ and R₂ are individually selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkenyls or C₂-C₁₀ heteroalkynyls and —(CR₁₅R₁₆)_(p)-D; wherein: R₁₅ and R₁₆ are individually selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; and C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkenyls or C₂-C₁₀ heteroalkynyls; p is a positive integer from 1 to about 12; D is selected from the group consisting of —SH, —OH, X₂, —CN, —OR₁₉, NHR₂₀,

wherein: R₁₇ is H, CH₃ or X₃; R₁₈ is H, a C₁₋₄ alkyl or benzyl; R₁₉ is H, a C₁₋₄ alkyl, X₂ or benzyl; R₂₀ is H, a C₁₋₁₀ alkyl or —C(O)R₂₁, wherein R₂₁ is H, a C₁₋₄ alkyl or alkoxy, t-butoxy or benzyloxy; X₂ and X₃ are independently selected halogens; R₃ is H, CH₃, or —C(═O)(CR₁₅R₁₆)_(w)-D, where w is 0 or an integer from 1 to about 12, and D is H or as described for R₁ and R₂; J is O, NH or S; R₄, R₅, and R₆ are independently selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls and halogens; Z is NR₇R₈ or

wherein R₇ is selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, alkenyls or alkynyls straight or branched; C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls, or —(CR₂₃R₂₄)_(q)-aryl, or R₈, wherein R₂₃ and R₂₄ are independently selected from the group consisting of H and C₁-C₁₀ alkyls; q is an integer from 1 to about 6; R₈ is selected from the group consisting of (CR₉R₁₀)_(n)-NR₂₂-R₁₁, (CR₉R₁₀)_(n)-CH₂-NHC(O)R₂₆ and (CR₉R₁₀)_(n)-CH₂-E; wherein R₉ and R₁₀ are independently selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkenyls or C₂-C₁₀ heteroalkynyls and halogens; R₂₆ is H, CH₃, O-t-butyl, O-benzyl; E is OH, SH or O—C(O)R₂₇, wherein R₂₇ is a C₁-C₆ alkyl, benzyl or phenyl; R₂₂ is H or CH₃; n is a positive integer from 1 to about 10; R₁₁ is H or-L-B, wherein L is a positive linker covalently linking NR₂₂ to B; and B is selected from the group consisting of polymers, biologically active materials and polymeric supports; R₂₅ is H, —C(O)—R₂₈ or —C(O)—O—R₂₉, wherein R₂₈ is a C₁-C₆ alkyl or benzyl; and R₂₉ is CH₃, t-butyl or benzyl; X₁ is O, NH, or S; and A is A₂ wherein X₁A₂ is an enzyme, or a pharmaceutical salt thereof.
 2. The compound of claim 1, wherein Z is NR₇R₈.
 3. The compound of claim 2, wherein R₈ is —CH₂—CH₂—NH₂.
 4. The compound of claim 2, wherein R₈ is (CR₉R₁₀)_(n)—NR₂₂—R₁₁.
 5. The compound of claim 1, wherein L-B is a maleimidyl or an N-hydroxysuccinimidyl compound.
 6. The compound of claim 4, wherein R₁₁ is a polyalkylene oxide residue.
 7. The compound of claim 6, wherein said polyalkylene oxide residue is a polyethylene glycol.
 8. The compound of claim 7, wherein said polyethylene glycol has a number average molecular weight of from about 2,000 to about 200,000 daltons.
 9. The compound of claim 4, wherein R₁₁ is a member of the group consisting of collagen, glycosaminoglycan, poly(-aspartic acid), poly(-L-lysine), poly(-lactic acid), poly-N-vinylpyrolidone and copolymers of poly(-lactic acid) and poly(-glycolic acid).
 10. The compound of claim 1, wherein R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from the group consisting of H, CH₃ and CH₃CH₂.
 11. The compound of claim 4, wherein R₇ is CH₃CH₂; R₈ is —(CR₉R₁₀)_(n)—NR₂₂—R₁₁; and R₉ and R₁₀ are H; n is 2; and X₁is O, S or NH.
 12. The compound of claim 4, wherein R₇ is CH₃CH₂; R₈ is —(CR₉R₁₀)_(n)—NR₂₂—R₁₁ and R₉ and R₁₀ are H.
 13. The compound of claim 1, wherein A₂ is an enzyme selected from the group consisting of serine proteases, cysteine proteases, esterases, lipases and enzymes containing an active-site serine or cysteine.
 14. The compound of claim 1, wherein J is O, R₂ is H, R₇ is CH₃CH₂; R₈ is -(CR₉R₁₀)_(n)-NR₂₂-R₁₁, R₉ and R₁₀ are H, and n is
 2. 15. The compound of claim 13, wherein X₁A₂ is an enzyme having an active-site serine or cysteine.
 16. The compound of claim 1, wherein X₁A₂ is a blood coagulation factor.
 17. The compound of claim 1, wherein the enzyme is selected from the group consisting of plasmins, urokinases, and tissue plasminogen activators.
 18. A compound of claim 1 selected from the group consisting of;


19. A method of treatment, comprising: administering to a mammal in need of such treatment an effective amount of a compound of formula (I)

wherein; R₁ and R₂ are individually selected from the group consisting of H, CH_(3.) C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls. straight or branched; C₂-C₁₀ heteroalkyls. C₂-C₁₀ heteroalkenyls or C₂-C₁₀ heteroalkynyls and -(CR₁₅R₁₆)_(p)-D; wherein: R₁₅ and R₁₆ are individually selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls straight or branched; and C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkenyls or C_(2-C) ₁₀ heteroalkynyls; p is a positive integer from 1 to about 12; D is selected from the group consisting of—SH, —OH, X₂, —CN, —OR₁₉, NHR₂₀,

wherein: R₁₇ is H, CH₃ or X₃; R₁₈ is H, a C₁₋₄ alkyl or benzyl; R₁₉ is H, a C₁₋₄ alkyl, X₂ or benzyl; R₂₀is H, a C₁₋₁₀ alkyl or —C(O)R₂₁, wherein R₂₁ is H. a C₁₋₄ alkyl or alkoxy, t-butoxy or benzyloxy; X₂ and X₃ are independently selected halogens; R₃ is H, CH₃, or —C(=O)(CR₁₅R₁₆)_(w) -D, where w is 0 or an inteiler from 1 to about 12, and D is H or as described for R₁ and R₂; J is O, NH or S; R₄, R₅, and R₆ are independently selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls. C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls and halogens; Z is NR₇R₈ or

wherein R₇ is selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, alkenyls or alkynyls straight or branched; C₂-C₁₀ heteroalkyls, heteroalkenyls, or heteroalkynvis, or -(CR₂₃R₂₄)_(q)-aryl, or R₈, wherein R₂₃ and R₂₄ are independently selected from the group consisting of H and C_(1-C) ₁₀ alkyls; q is an integer from 1 to about 6; R₈ is selected from the group consisting of (CR₉R₁₀)_(D)-NR₂₂-R₁₁, (CR₉R₁₀ )_(n)-CH₂-NHC(O)R₂₆ and (CR₉R₁₀)_(n)-CH₂-E; wherein R₉ and R₁₀ are independently selected from the group consisting of H, CH₃, ₂-C₁₀alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkynyls and halogens; R₂₆ is H, CH₃, O-t-butyl, O-benzyl; E is OH, SH or O—C(O)R₂₇, wherein R₂₇ is a C₁-C₆ alkyl, benzyl or phenyl; R₂₂ is H or CH₃; n is a positive integer from 1 to about 10; R₁₁ is H or -L-B, wherein L is a linker covalently linking NR₂₂ to B; and B is selected from the group consisting of polymers. biologically active materials and polymeric supports; R₂₅ is H, —C(O)-R₂₈ or —C(O)-R₂₉, wherein R₂₈ is a C₁₋C₆ alkyl or benzyl; and R₂₉ is CH₃, t-butyl or benzyl; X₁ is O,NH, or S; and A is A₂ wherein X₁A₂ is an enzyme, or a pharmaceutical salt thereof.
 20. The method of claim 19, further comprising exposing the compound of formula (I) to an energy source after administration to said mammal.
 21. The method of claim 20, wherein the energy source is white light having a wavelength in the range from 340 to 700 nm.
 22. The method of claim 20, wherein the energy source is white light having a wavelength in the range from 350-420 nm.
 23. The method of claim 20, wherein the energy source is selected from the group consisting of microwave, ultrasound, radio energy, gamma radiation, radioactivity, ultraviolet light and infrared light.
 24. A method of preparing a conjugate, comprising; reacting a compound of Formula (IV)

wherein: R₁ and R₂ are individually selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkenyls or C₂-C₁₀ heteroalkynyls and —(CR₁₅R₁₆)_(p)-D wherein: R₁₅ and R₁₆ are individually selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls and C₂-C₁₀ alkynyls, straight or branched; and C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroalkenyls or C₂-C₁₀ heteroalkynyls; p is a positive integer from 1 to about 12; D is selected from among —SH, —OH, X₂, —CN, —OR₁₉, NHR₂₀,

wherein: R₁₇ is H, a CH₃ or X₃; R₁₈ is H, a C₁₋₄ alkyl or benzyl; R₁₉ is H, a C₁₋₄ alkyl, X₂ or benzyl; R₂₀ is H, a C₁₋₁₀ alkyl or —C(O)R₂₁ wherein R₂₁ is H, a C₁₋₄ alkyl or alkoxy, t-butoxy or benzyloxy; X₂ and X₃ are independently selected halogens; R₃ is H, CH₃, or —C(═O)(CR₁₅R₁₆)_(w)-D, where w is 0 or an integer from 1 to about 12, and D is H or as described for R₁ and R₂, J is O, NH or S; R₄, R₅ and R₆ independently selected from the group consisting of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkenyls or C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, heteroalkenyls or heteroalkynyls and halogens; R₇ is selected from among H, CH₃ and C₂-C₁₀ alkyls; R₉ and R₁₀ are independently selected from the group of H, CH₃, C₂-C₁₀ alkyls, C₂-C₁₀ alkynyls, straight or branched; C₂-C₁₀ heteroalkyls, C₂-C₁₀ heteroakynyls, and halogen; n is a positive integer from 1 to 10; X₁ is O, NH, or S; R₂₂ is H or CH₃; and A is A₂ wherein X₁A₂ is an enzyme with a compound of the Formula (V): L₁−B₁   (V) wherein L₁ is a moiety containing a functional group capable of reacting with the NHR₂₂ of Formula (IV); and B₁ is selected from the group consisting of polymers, biologically active materials and polymeric supports.
 25. The method of claim 24, wherein L₁-B₁ includes a member selected from the group consisting of heterobifunctional reagents having N-hydroxysuccinimide and maleimide, bifunctional maleimide and bifunctional polyethylene glycol.
 26. The method of claim 25, wherein the heterobifunctional reagent is Succinimidyl-6-[(β-maleimidopropionamido) hexanoate].
 27. The compound of claim 1, wherein L-B is selected from the group consisting of heterobifunctional reagents having N-hydroxysuccinimide and maleimide, bifunctional maleimide and bifunctional polyethylene glycol.
 28. The compound of claim 7, wherein said polyethylene glycol is bifunctional polyethylene glycol.
 29. The compound of claim 16, wherein X₁A₂ is selected from the group consisting of thrombin (Factor IIa), Factor VIIa, Factor IXa, Factor Xa, Factor XIa and Factor XIIa.
 30. The compound of claim 1, wherein L is a linker selected from the group consisting of succinimides, maleimides, imidoesters, 2-iminothiolanes, hydrazides, maleic anhydrides, azides, citraconic anhydrides and glutaraldehydes.
 31. The compound of claim 1, wherein B is selected from the group consisting of antibodies, fragments thereof, single chain binding antibodies, monoclonal antibodies, growth factors, interferons, cytokines, cell-surface binding metabolites, lectin, sugar-peptide, sugar-petide-lipid targeting agents, proteins, nucleic acids, lectins, lipids, carbohydrates, inhibitors of cell surface enzymes, PAOs, glycosaminoglycans, dextran, poly-glutamic acid, poly-aspartic acid, poly-L-lysine and mixtures thereof, polyvinylpyrrolidine, collagen, peptides, hormones, ligands for receptors, and carriers and supports in form of columns, films, membranes, beads, particles, microparticles, and filters. 